Development of the ovarian follicle is a complex process integral to female fertility. The objective of this proposal is to expand understanding of the mechanism(s) causing the formation of aberrant multi-oocytic follicles (MOFs) during vertebrate folliculogenesis using a novel study species, the American alligator. Multioocytic follicles (MOFs, also called polyovular follicles) have been associated with decreased success rates of in vitro fertilization and increased embryonic loss. MOFs have been studied in laboratory animals, however, a natural population of alligators has been identified in which 100% of neonatal females exhibit this pathology apparently due to environmental contaminant exposure. In comparison, reference alligator populations exhibit no MOFs or a very low frequency. Due to the conserved nature of folliculogenesis, we propose this comparative approach to address the mechanism(s) associated with the development of MOFs. Laboratory research has identified both estrogens and the transforming growth factor inhibin as factors that can induce MOF formation. The central hypothesis of this proposal is that the disruption of the ovarian hormonal milieu by exogenous factors alter ovarian follicle development leading to MOFs. The studies proposed will test this hypothesis by investigating 3 related specific aims. Aim 1 will examine the gene expression patterns for selective components of the estrogen and inhibin/activin signaling pathways in cellular compartments of normal and multioocytic ovarian follicles using laser capture microdissection and quantitative RT-PCR. Aim 2 will examine estrogen-induced perturbation of the inhibin A/Activin A signaling pathway and their potential role in altered granulosa cell proliferation or apoptosis. This will be tested by embryonic exposure to estradiol followed by FSH challenge during 2 life stages. Aim 3 will examine environment estrogen or pharmaceutical anti-estrogen-induced perturbation of the inhibin A/Activin A signaling pathway and their role in altered granulosa cell proliferation or apoptosis to firmly establish if an environmental perturbation leads to altered inhibin/activin signaling. In total, the studies described in this application provide a novel approach to provide new insights into normal follicle development, establish mechanisms by which normal development could be disrupted leading to aberrant ovarian pathologies such as multi-oocytic follicles, and offer new insights into the regulatory functions of inhibin/activins and estrogens in this important reproductive process. Understanding and documenting the conserved mechanisms of induction of MOFs among vertebrates will strengthen the causal and mechanistic relationships, allowing a better understanding of this phenomenon in human populations. [unreadable] [unreadable] [unreadable]